Traditional plant breeding is based on repeated cross-breeding of plant lines individually carrying desired qualities. The identification of progeny lines carrying all the desired qualities is a particularly time-consuming process as the biochemical and genetic basis of the qualities is usually unknown. New lines are therefore chosen according to their phenotype, usually after a screening of many lines in field experiments.
Through the ages a direct connection has existed between the stage of nutrition, i.e. the health, of the population and the agricultural possibility of ensuring a sufficient supply of nitrogen in order to obtain satisfactory yields. Already in the seventeenth century it was discovered that plants of the family leguminosae including beyond peas also beans, lupins, soybean, bird's-foot trefoil, vetches, alfalfa, sainfoin, and trefoil had an ability of improving crops grown on the habitat of these plants. Today it is known that the latter is due to the fact that the members of the plants of the family leguminosae are able to produce nitrogen reserves themselves. On the roots they carry bacteria with which they live in symbiosis.
An infection of the roots of these leguminous plants with Rhizobium bacteria causes a formation of root nodules able to convert atmospheric nitrogen into bound nitrogen, which is a process called nitrogen fixation.
Atmospheric nitrogen is thereby converted into forms which can be utilized by the host plant as well as by the plants later on growing on the same habitat.
In the nineteenth century the above possibility was utilized for the supply of nitrogen in order to achieve a novel increase of the crop yield.
The later further increases in the yield have, however, especially been obtained by means of natural fertilizers and nitrogen-containing synthetic fertilizers. The resulting pollution of the environment makes it desirable to provide alternative possibilities of ensuring the supply of nitrogen necessary for the best possible yields obtainable.
It would thus be valuable to make an improvement possible of the existing nitrogen fixation systems in leguminous plants as well as to allow an incorporation of nitrogen fixation systems in other plants.
The recombinant DNA technique and the plant transformation systems developed render it now possible to provide plants with new qualities in a well-controlled manner. These characteristics can derive from not only the same plant species, but also from all other procaryotic or eucaryotic organisms. The DNA techniques allow further a quick and specific identification of progeny lines carrying the desired qualities. In this manner a specific plant line can be provided with one or more desired qualities in a quick and well-defined manner.
Correspondingly, plant cells can be provided with well-defined qualities and subsequently be maintained as plant cell lines by means of known tissue culture methods. Such plant cells can be utilized for the production of chemical and biological products of particular interest such as dyes, flavours, aroma components, plant hormones, pharmaceutical products, primary and secondary metabolites as well as polypeptides (enzymes).
A range of factors and functions necessary for biological production of a predetermined gene product are known. Both the initiation and regulation of transcription as well as the initiation and regulation of posttranscriptional processes can be characterised.
At the gene level it is known that these functions are mainly carried out by 5' flanking regions. A wide range of 5' flanking regions from procaryotic and eucaryotic genes has been sequenced, and in view inter alia thereof a comprehensive knowledge has been provided of the regulation of gene expression and of the sub-regions and sequences being of importance for the regulation of expression of the gene. Great differences exist in the regulatory mechanism of procaryotic and eucaryotic organisms, but many common features apply to the two groups.
The regulation of the expression of gene may take place on the transcriptional level and is then preferably exerted by regulating the initiation frequency of transcription. The latter is well known and described inter alia by Benjamin Lewin, Gene Expression, John Wiley & Sons, vol. I, 1974, vol. II, Second Edition 1980, vol. III, 1977. As an alternative the regulation may be exerted at the posttranscriptional level, e.g. by the regulation of the frequency of the translation initiation, at the rate of the translation, and of the termination of the translation.
It has been shown in connection with the present invention that 5' flanking regions of root nodule-specific genes, exemplified by the 5' flanking region of the soybean leghemoglobin Lbc.sub.3 gene, can be used for inducible expression of a foreign gene in an alien leguminous plant. The induction and regulation of the promoter is preferably carried out in the form of a regulation and induction at the transcriptional level and differs thereby from the inducibility stated in Danish patent application No. 4889/85, the latter inducibility preferably being carried out at the translation level.
The transcription of both the Lbc.sub.3 gene of the soybean and of the chimeric Lbc.sub.3 gene transferred to bird's-foot trefoil starts at a low level immediately upon the appearance of the root nodules on the plant roots. Subsequently, a high increase of the transcription takes place immediately before the root nodules turn red. The transcription of a range of other root nodule-specific genes is initiated exactly at this time. The simultaneous induction of the transcription of the Lb genes and other root nodule-specific genes means that a common DNA sequence(s) must be present for the various genes controlling this pattern of expression. Thus the leghemoglobin-c.sub.3 gene is a representative of one class of genes and the promoter and the leader sequence, target areas for activation as well as the control elements of the tissue specificity of the Lbc.sub.3 gene are representatives of the control elements of a complete gene class.
The promoter of the 5' flanking regions of the Lb genes functions in soybeans and is responsible for the transcription of the Lb genes in root nodules. It is furthermore known, that the efficiency of both the transcription initiation and the subsequent translation initation on the leader sequence of the Lb genes is high as the Lb proteins constitute approximately 20% of the total protein content in root nodules.
The sequence of 5' flanking regions of the four soybean leghemoglobin genes Lba, Lbc.sub.1, Lbc.sub.2, and Lbc.sub.3 appears from the enclosed sequence scheme, scheme 1, wherein the sequences are stated in such a manner that the homology between the four 5' flanking regions appears clearly.
In the sequence scheme "-" indicates that no base is present in the position in question. The names of the genes and the base position counted upstream from the ATG start codon are indicated to the right of the sequence scheme. Furthermore the important sequences have been underlined.
As its appears from the sequence scheme a distinct degree of homology exists between the four 5' flanking regions, and in the position 23-24 bp upstream from the CAP addition site they all contain a TATATAAA sequence corresponding to the "TATA" box which in eukaryotic cells usually are located a corresponding number of bp upstream from the CAP addition site. Furthermore a CCAAG sequence is present 64-72 bp upstream from the CAP addition site, said sequence corresponding to the "CCAAT" box usually located 70-90 bp upstream from the CAP addition site. From the CAP addition site to the translation start codon, ATG, leader sequences of 52-69 bp are present and show a distinct degree of homology of approx. 75-80%.
In accordance with the present invention it has furthermore been proved, exemplified by Lbc.sub.3, that the 5' flanking regions of the soybean leghemoglobin genes are functionally active in other plant species. The latter has been proved by fusing the E. coli chloroamphenicol acetyl transferase (CAT) gene with the 5' and 3' flanking regions of the soybean Lbc.sub.3 gene in such a manner that the expression of the CAT gene is controlled by the Lb promoter. This fusion fragment was cloned into the integration vectors pAR1 and pAR22, whereby the plasmids pAR29 and pAR30 were produced. Through homologous recombination the latter plasmids were integrated into the Agrobacterium rhizogenes T DNA region. The transformation of Lotus corniculatus (bird's-foot trefoil) plants, i.e. transfer of the T DNA region, was obtained by wound infection on the hypocotyl. Roots developed from the transformed plant cells were taken in in vitro culture and freed from A. rhizogenes bacteria by means of antibiotics. Completely regenerated plants were produced by these root cultures through somatic embryogenesis or organogenesis.
Regenerated plants were subsequently inoculated with Rhizobium loti bacteria and root nodules for analysis were harvested. Transcription and translation of the chimeric Lbc.sub.3 CAT gene could subsequently be detected in root nodules on transformed plants as the activity of the produced chloroamphenicol acetyl transferase enzyme.
The conclusion can subsequently be made that the promoter-containing 5' flanking regions of root nodule-specific genes exemplified by the soybean Lbc.sub.3 promoter are functionally active in foreign plants. The latter is a surprising observation as root nodules are only developed as a consequence of a very specific interaction between the leguminous plant and its corresponding Rhizobium microsymbiont.
Soybeans produce nodules only upon infection by the species Rhizobium japonicum and Lotus corniculatus only upon infection by the species Rhizobium loti. Soybean and Lotus corniculatus belong therefore to two different cross-inoculation groups, each group producing root nodules by means of two different Rhizobium species. The expression of a chimeric soybean gene in Lotus corniculatus proves therefore an unexpected universal regulatory system applying to the expression of root nodule-specific genes. The regulatory DNA sequences involved can be placed on the 5' and 3' flanking regions of the genes, here exemplified by the 2.0 Kb 5' and 0.9 Kb 3' flanking regions of the Lbc.sub.3 gene. This observation allows the use of root nodule-specific promoters and regulatory sequences in any other plant species and any other plant cell line.